In eukaryotic cells, one of the important mechanisms of gene regulation is the modulation of transcription. Eukaryotic nuclei possess three distinct systems to accomplish this, each acting on a different set of cellular genes. To date only two of these systems have been accessible to biochemical study. This proposal is directed toward filling this gap by developing an in vitro transcription system for ribosomal RNA genes. Three components are required to elicit selective transcription in vitro: RNA polymerase I, a recombinant DNA template and one or several transcription factors (TF) to direct interaction of the polymerase with the DNA. The first two of these have been produced and characterized previously. The major thrust of this study is the identification, purification and characterization of the TFs followed by studies of their role in regulation of ribosomal RNA synthesis. Development of a complementation assay for the TFs is proposed, followed by purification of the factors to homogeneity by standard protein purification techniques. The basic structure of the TFs, their possible modification as part of a regulatory network and the number of molecules of each TF in cells undergoing regulation of the transcription unit will be studied using electrophoretic techniques coupled with immunological probes. Since transcription of ribosomal RNA is regulated in concert with the growth-division rate of cells, this project will not only complete the triad of transcriptional systems available for biochemical scrutiny, but may also shed some light on the mechanisms regulating cellular growth.